The application of mycotoxin genotyping GSK1120212 assays reveals the toxigenic potential of fungal strains from pure cultures and predicts the presence of certain compounds in tested plant material (Niessen, 2008).
A multiplex PCR assay based on homologues of the esyn1 gene has been developed for the detection of Fusarium spp. with the potential to produce enniatins (Kulik et al., 2007). However, this assay is qualitative and requires the time-consuming identification of expected PCR products on ethidium bromide-stained agarose gels (end-point PCR). The aim of this study was to develop an alternative, quantitative TaqMan MGB (Minor Groove Binder) assay based on esyn1 homologues present in the genomes of F. avenaceum/F. tricinctum and F. poae, respectively. The assays developed were tested on asymptomatic wheat grain samples in relation to enniatins levels. One hundred and eleven fungal isolates used for the specificity of TaqMan assays are listed in Table 1.
The strains of Fusarium tested are held in the CBS Selleckchem 17-AAG (CBS Fungal Biodiversity Centre, Utrecht, the Netherlands) fungal collection. IBT isolates were kindly provided by Dr Ulf Thrane (Department of Systems Biology, Center for Microbial Biotechnology, Technical University of Denmark). Polish field isolates [Department of Diagnostics & Plant Pathophysiology (DDPP)] were obtained from 155 wheat seed samples that were collected randomly from fields located in different parts of Poland (data not shown). All isolates used this study are held in the fungal collection of the DDPP (University of Warmia and Mazury, Olsztyn, Poland). The Fusarium isolates were maintained on potato dextrose agar at 25 °C before DNA extraction. Asymptomatic wheat seed samples (48 × 1 kg) were harvested in 2007 and 2008 from 45 fields located in northern Poland. Enniatins were analyzed as described in Jestoi et al. (2005). In brief, 25 g of ground grain samples were extracted with 84% acetonitrile. The raw extract was purified using
a C8-solid phase extraction. Enniatins were determined with HPLC combined with a tandem mass spectrometer (LC-MS/MS) by detecting specific product ions for each of the analytes. The limits of quantifications were 0.6, 4, 3.8 and 10.8 μg kg−1 Baf-A1 for enniatin A, enniatin A1, enniatin B and enniatin B1, respectively. DNA extraction from the grain as well as from fungal cultures was carried out as described previously (Kulik et al., 2007). A fragment of the esyn1 homologue of 40 F. poae isolates isolated from different hosts and geographical locations (data not shown) was amplified with the primers Esy1 ttc aag ggc tgg acg tct atg and Esypoae2 cag cat atc gat acg cgc tga g, designed on the basis of a conserved region of published sequence data of Fusarium species deposited in the NCBI database. The amplified product was sequenced in both directions using the above primers.